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Use-Dilution Test: Definition & Uses

Instructor: Hannah Hart

Biology Expert Q&A Copywriter (Contract) for Study.com

Explore the use-dilution test, its history, and its application in measuring the effectiveness of various disinfectants in reducing microbial counts on hard surfaces. Updated: 07/01/2022

Definition and Background of the Use-Dilution Test

The use-dilution test is a method of analyzing the efficacy of a liquid disinfectant in decreasing microbial counts on hard, nonporous, inanimate surfaces in the environment. A small stainless steel cylinder, usually measuring around 10 mm in length and 8 mm in diameter, is coated in a culture of a specific microorganism and allowed to dry. When the cylinder is dry, it is dipped in a series of disinfectant solutions for set amounts of time. The cylinder is then placed in a test tube of sterile medium and allowed to incubate. If microbes are present on the cylinder, the medium will appear turbid (cloudy), indicating that the disinfectant was ineffective at significantly reducing microbial numbers. If the medium appears clear following incubation, the disinfectant is likely effective at markedly reducing microbial numbers. Developed by the Association of Official Agricultural Chemists International (AOAC), a nonprofit group that sets many protocol standards in the field of chemistry, the use-dilution test must usually produce positive results in 59 out of 60 replications to establish a valid claim of the efficacy of the disinfectant, across multiple batches of disinfectant and multiple days of tests.

Applications of the Use-Dilution Test

The AOAC standards as described above have been adopted by numerous entities, from government agencies to private companies, for determining appropriate disinfectants to use in a variety of settings.

The Use-Dilution Test in Government Agencies

In 2020, the Environmental Protection Agency (EPA) and its Office of Pesticide Programs published a revised SOP (standard operating procedure) document describing the use-dilution method in testing disinfectants against three major microbes that commonly form biofilms (a group of bacteria that adhere together and to a surface): Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella enterica. The EPA uses the use-dilution test to assess the efficacy of antimicrobial pesticides on these biofilm-forming bacteria.

The Use-Dilution Test in Research

The use-dilution test can also be used to test disinfectants on viral pathogens, such as the Newcastle Disease Virus, which causes the reportable, contagious, and often fatal Newcastle Disease in poultry such as chickens. Finding an effective disinfectant for use in livestock settings such as a large layer or broiler farms is essential to inactivating any virus in the environment and preventing Newcastle Disease from gaining a foothold in the poultry industry. In 1964, researchers from the University of California modified the original use-dilution test, dating back to 1960, for use on viral microorganisms. They were able to produce useful results and determine that use-dilution tests are valuable for testing disinfectants on viruses and bacteria.

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